Studies of the Ligand Binding Site in the 5-HT 3 Receptor
2.4 A Hydrogen Bond in Loop A Is Critical for Binding and Function of the 5-HT 3 Receptor
2.4.3 Discussion
expressed in HEK cells (43). For Asn128 mutant receptors there was no significant differences in [3H]granisetron binding affinity for any substitution studied, while no specific binding was observed for any Glu129 mutant receptor, at concentrations up to 20 nM. In the current study we examined single point [3H]granisetron binding to solubilized oocyte preparations. No specific radioligand binding was observed at 0.5 nM
[3H]granisetron for E129A, E129G and E129K mutant receptors, while levels of binding in Asn128 receptors were similar to WT receptors (figure 2.19). These data suggest that Glu129 substitutions ablate high affinity antagonist binding, but at least some
substitutions permit agonist binding, as large (> 5 µA) responses to 5-HT and mCPBG were observed for E129D and E129N receptors.
holds the channel closed in the absence of agonists, and reduces the probability of channel opening (57). More recent studies, in particular the high resolution structure determination of AChBP, confirm the importance of the loop A Tyr at position 89, (equivalent to Tyr93 in nAChR) which is in close contact with bound ligands (61). The aligned Tyr is also important in GABAA receptors; Tyr97 in the β2 subunit has recently been shown to make a cation-π interaction with GABA (62). It was therefore not surprising that the aligning residue in the 5-HT3 receptor, Asn128, was considered to be important. Homology modeling identified it as the only loop A residue in the binding pocket, and predicted a hydrogen bond between Asn128 and 5-HT (15). However, experimental studies have cast some doubt on this conclusion, as changing Asn128 did not affect [3H]granisetron binding affinity (43).
Our new data, incorporating both natural and unnatural amino acids at this position, provide a detailed analysis of the role of Asn128, and strongly suggest that Asn128 has its most significant role in the conformational change that results in receptor gating. All Asn128 mutant receptors showed changes in their functional characteristics (figure 2.15b), but these were particularly evident in N128Q receptors. Gln has similar chemical properties to Asn, yet this mutation markedly slows apparent current activation, increases the relative efficacy (Rmax) of the partial agonist mCPBG, and eliminates receptor desensitization (figures 2.15 and 2.17). Changes in current activation and Rmax strongly suggest effects on receptor opening, and, while desensitization is not well understood, it is known to be influenced by channel opening and closing rates, and the rates of conformational changes to and from the desensitized state. These observations
therefore all suggest that Asn128 has a role in facilitating transitions between conformational states rather than direct effects on ligand binding. In the new model this residue is close to loop B, especially Thr179, and both these residue contribute to a complex network of hydrogen bonds that could potentially be involved in the conformational change that results in receptor gating.
Receptors with substitutions at Glu129 have, in the past, been insufficiently characterized due to problems with low expression (43,55). In this study, these problems have been largely overcome by the use of Xenopus oocytes as expression hosts. Large responses to 5-HT and the partial agonists mCPBG and 5-FT were measured with mutants of Glu129 that did not previously display measurable currents when expressed in HEK293 cells. Interestingly, only the Glu129 mutant receptors where Glu was replaced with residues that have the ability to accept a hydrogen bond responded robustly to 5-HT application, suggesting that this property is critical for 5-HT binding. Previous ligand docking data has indicated that the hydroxyl of 5-HT is located in this region of the binding pocket, and in the new model this hydroxyl would donate a hydrogen bond to Glu129; more specifically one of the side chain oxygens (Os) of Glu129 would interact with the hydrogen of the 5-HT 5-hydroxyl (figure 2.20). Note that an ionic interaction involving Glu129 is not supported by our data with the unnatural amino acid Nha. This amino acid is structurally similar to Glu: the nitro group is planar, like the carboxylate, and the two N-O bonds are of equal length, as are the C-O bonds in carboxylate. Two resonance structures are possible (as with carboxylate) but in a nitro group the nitrogen (N) carries a positive charge and the Os share a negative charge—thus overall the group
is neutral, in contrast to the negative charge on a carboxylate; a nitro group could therefore not contribute to an ionic bond. As there was no significant increase in EC50
when Nha was substituted for Glu, it shows that an ionic bond is not formed here. Nha could, however, still form a hydrogen bond as each O in the nitro group has two lone pairs of electrons (as does the carboxylate), which can serve as hydrogen bond acceptors.
Interestingly, mutations at Glu129 have no effect on the EC50s of the partial agonists mCPBG or 5-FT (figure 2.21). This might be expected with mCBPG, which has a distinct structure to 5-HT and is unlikely to interact with identical binding site residues, but the only difference between 5-HT and 5-FT is the group at the 5 position. The OH of
Figure 2.20. The new model of 5-HT3 receptor binding site, showing 5-HT hydrogen bonded to Glu129. This model is that described by Sullivan et al. 2006, where a single amino acid gap was inserted into the 5-HT3A receptor subunit sequence (accession number: Q6J1J7) following V131 (WVPDILINEFV-DVG). The new model of the complete mouse 5-HT3A receptor extracellular domain was then built using L. stagnalis AChBP (accession number P58154, PDB ID 1I9B) as a template. The locations of Asn128, Glu129 and Trp183 relative to 5-HT are shown. The proposed H- bond between Glu129 and the hydroxyl group of 5-HT is shown in green.
5-HT is a good hydrogen bond donor and a moderately good hydrogen bond acceptor;
however the F of 5-FT cannot donate a hydrogen bond and is a very poor hydrogen bond acceptor. Thus if 5-FT binding in the same orientation as 5-HT, which seems likely, it is probable that there is no hydrogen bond here with Glu129, a hypothesis that is supported by the data. The lack of this bond may be the reason why 5-FT only acts as a partial agonist.
If Glu129 interacts directly with 5-HT, then it must face into the binding site and could interact with antagonists. Our, and previous, data support this proposal: there is no specific [3H]granisetron binding to Glu129 mutant receptors in either HEK cells or oocyte membranes in the usual subnanomolar range (13). Interestingly, though, granisetron does appears to be able to bind to E129D mutant receptors at higher concentrations, as 10 nM granisetron inhibited ~80% of 5-HT-induced currents ( WT IC50
= 0.2 nM; (63)). Combined with the fact that E129D mutant receptors recover more quickly than WT receptors from granisetron inhibition, these data suggest that E129D mutant receptors have a higher dissociation rate constant for granisetron. Such an explanation is consistent with previous equilibrium radioligand binding studies, where an
Figure 2.21. Comparison between agonists on the effect of mutation at Glu 129. mCPBG and 5-FT, which lack hydroxyl groups show little change in EC50.
~100-fold decrease in the granisetron Kd was reported (9).
Our data also reveal small but significant changes in relative efficacies for mCPBG at functional Glu129 mutant receptors, indicating there may also be a role for this residue in the conformational changes leading to receptor gating. These changes are opposite to those we observed with Asn128. We do not yet understand what this implies, although it may be related to the different roles of the 2 residues and/or distinct mechanisms of action or critical binding residues used by different agonists. In support of this latter hypothesis, a similar study on a series of loop C residues, which are also proposed to play a role in binding and/or gating, revealed increases in mCPBG efficacy but decreases in the efficacy of another partial agonist, 2-methyl-5-HT, in the same mutant receptors (64). In our study, the conversion of mCPBG from a partial agonist to an antagonist at E129Q mutant receptors could reflect a change in affinity of mCPBG for certain conformational states of the receptor only (e.g., a reduction in affinity of the open state but not the closed state). This would correspond to the ‘K’ phenotype of allosteric receptor mutants described by Galzi et al. (65).
The importance of Glu129 suggests it may be equivalent to Tyr93 in the nACh receptor, which has also been proposed to play a role in both binding and function.
Mutating Tyr93 results in a rightward shift of the dose-response curve (66), mainly because of slower ligand-association and channel-opening rate constants (67). Similarly, the equivalent residue in the GABAA receptor, β2Tyr97, which directly contacts GABA through a cation-π interaction (62), may also be involved in gating; mutation to Cys
causes spontaneous activation (56). Aligning Glu129 and Tyr93 requires that a space be inserted in the conserved WxPDxxxN domain in loop A of the nACh receptor. This sequence is critical for locating the B loop in the nACh receptor through interactions involving Asp89 (68). More recent data, however, show that in non-ACh receptors the xxxN portion of this region may not be critical; in the GABAA receptor, for example, two amino acid ‘spaces’ must be inserted in the ‘xxx’ tract to allow β2Tyr97 to contribute to the binding pocket. We therefore propose that Glu129 is equivalent to Tyr93, and faces into the binding pocket, where it forms a hydrogen bond with the 5-OH group of 5-HT.
2.5 Structure-function Studies on the 5-HT3 Receptor Ligand